Nanoflow gradient generator for capillary high-performance liquid chromatography

A novel method of generating a nanoflow gradient elution for a capillary high-performance liquid chromatography (HPLC) system has been developed. An important feature of this system is that any gradient (GR) profile generated by a conventional microflow GR pump can be asymptotically traced and converted as a corresponding nanoflow GR profile simply by using a 10-port switching valve with two injection loops installed. Consequently, it has been called an "asymptotic trace 10-port valve" (AT10PV) nanoflow GR generator. Performance of the AT10PV nanoflow GR generator was tested in the range of flow rates from 50 to 500 nL/min. The test demonstrated that the AT10PV nanoflow GR generator can asymptotically trace the original gradient profile with good reproducibility. A capillary HPLC system using the AT10PV nanoflow GR generator provides reasonably good repeatability of peak retention times on the chromatogram of the tryptic digest of a BSA sample, RSD of less than 0.3% at a flow rate of 200 nL/min. It also enables sequential running of a series of sample injections in the same manner as conventional analysis at microflow rates.     

Continuous pH/salt gradient and peptide score for strong cation exchange chromatography in 2D-nano-LC/MS/MS peptide identification for proteomics

Tryptic digests of human serum albumin and human lung epithelial cell lysates were used as test samples in a novel proteomics study. Peptides were separated and analyzed using 2D-nano-LC/MS/MS with strong cation exchange (SCX) and reversed-phase chromatography and continuous gradient elution. The peptide elution conditions combined simultaneous pH gradient with ammonium acetate salt gradient elution modes. A novel empirical SCX peptide elution score was developed, which accounts for both the number of basic and acidic residues and, in part, their location within a sequence of a peptide. Average scores calculated for the fractionated peptide sequences correlated well with the pH of SCX elution fractions. Multiple peptides with identical amino acid sequences, but differing in cysteine tags possessing different positive charge and different SCX elution properties, were obtained by subjecting the samples to reduction and alkylation with different cysteine alkylating reagents: iodoacetamide, 4-vinylpyridine, and (3-acrylamidopropyl) trimethylammonium chloride. The structurally similar peptides were used as elution standards.     

Very high pressure gradient LC/MS/MS

A very high pressure liquid chromatography (VHPLC) system was constructed by modifying a commercially available pump in order to achieve pressures in excess of 1,200 bar (17,500 psi). A computer-controlled low-pressure mixer was used to generate solvent gradients. Protein digests were rapidly analyzed by reversed-phase VHPLC with linear solvent gradients coupled to either a tandem mass spectrometer using electrospray ionization or a UV/visible detector. The separations were performed at pressures ranging from 790 (11,500 psi) to 930 bar (13,500 psi) in 22-cm-long capillary columns packed with C18-modified 1.5-microm nonporous silica particles. A digest of bovine serum albumin (BSA) was analyzed by the VHPLC system connected to a mass spectrometer in MS mode. An analysis of 12.5 fmol of sample gave signal-to-noise ratios of tryptic peaks greater than 10:1 in the base peak plot mass chromatogram. This system was also used to analyze a proteolytic digest of a rat liver protein excised from a 2-D gel separation of a liver tissue lysate. For this analysis, the mass spectrometer was set up to perform data-dependent scanning (automated switching from MS mode to MS/MS mode when a peak was detected) for peptide sequencing and protein identification by database searching. The results of this analysis are compared to an analysis performed on the same sample using the nanoelectrospray-MS/MS technique. Though both techniques were able to identify the unknown protein, the VHPLC method gave twice as many sequenced peptides as nanoelectrospray and improved the signal-to-noise ratio of the spectra by at least a factor of 10. Direct comparisons with nanoelectrospray for MS and MS/MS data acquisition from a BSA digest were made. These comparisons show enhancements of greater than 20-fold for VHPLC over nanoelectrospray. In addition, the VHPLC/MS/MS data acquisition was accomplished in an automated manner.     

Open tubular immobilized metal ion affinity chromatography combined with MALDI MS and MS/MS for identification of protein phosphorylation sites

Protein phosphorylation is one of the most important known posttranslational modifications. Tandem mass spectrometry has become an important tool for mapping out the phosphorylation sites. However, when a peptide generated from the enzymatic or chemical digestion of a phosphoprotein is highly phosphorylated or contains many potential phosphorylation residues, phosphorylation site assignment becomes difficult. Separation and enrichment of phosphopeptides from a digest mixture is desirable and often a critical step for MS/MS-based site determination. In this work, we present a novel open tubular immobilized metal ion affinity chromatography (OT-IMAC) method, which is found to be more effective and reproducible for phosphopeptide enrichment, compared to a commonly used commercial product, Ziptip from Millipore. A strategy based on a combination of OT-IMAC, sequential dual-enzyme digestion, and matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight tandem mass spectrometry for phosphoprotein characterization is presented. It is shown that MALDI MS/MS with collision-induced dissociation can be very effective in generating fragment ion spectra containing rich structural information, which enables the identification of phosphorylation sites even from highly phosphorylated peptides. The applicability of this method for real world applications is demonstrated in the characterization and identification of phosphorylation sites of a Na(+)/H(+) exchanger fusion protein, His182, which was phosphorylated in vitro using the kinase Erk2.     

Single peptide-based protein identification in human proteome through MALDI-TOF MS coupled with amino acids coded mass tagging

Identification of proteins with low sequence coverage using mass spectrometry (MS) requires tandem MS/MS peptide sequencing. It is very challenging to obtain a complete or to interpret an incomplete tandem MS/MS spectrum from fragmentation of a weak peptide ion signal for sequence assignment. Here, we have developed an effective and high-throughput MALDI-TOF-based method for the identification of membrane and other low-abundance proteins with a simple, one-dimensional separation step. In this approach, several stable isotope-labeled amino acid precursors were selected to mass-tag, in parallel, the human proteome of human skin fibroblast cells in a residue-specific manner during in vivo cell culturing. These labeled residues can be recognized by their characteristic isotope patterns in MALDI-TOF MS spectra. The isotope pattern of particular peptides induced by the different labeled precursors provides information about their amino acid compositions. The specificity of peptide signals in a peptide mass mapping is thus greatly enhanced, resolving a high degree of mass degeneracy of proteolytic peptides derived from the complex human proteome. Further, false positive matches in database searching can be eliminated. More importantly, proteins can be accurately identified through a single peptide with its m/z value and partial amino acid composition. With the increased solubility of hydrophobic proteins in SDS, we have demonstrated that our approach is effective for the identification of membrane and low-abundant proteins with low sequence coverage and weak signal intensity, which are often difficult for obtaining informative fragment patterns in tandem MS/MS peptide sequencing analysis.     

Use of peptide retention time prediction for protein identification by off-line reversed-phase HPLC-MALDI MS/MS

A new algorithm, sequence-specific retention calculator, was developed to predict retention time of tryptic peptides during RP HPLC fractionation on C18, 300-A pore size columns. Correlations of up to approximately 0.98 R2 value were obtained for a test library of approximately 2000 peptides and approximately 0.95-0.97 for a variety of real samples. The algorithm was applied in conjunction with an exclusion protocol based on mass (15 ppm tolerance) and retention time (2-min tolerance for 0.66% acetonitrile/min gradient), MART criteria to significantly reduce the instrument time required for complete MS/MS analysis of a digest separated by RP HPLC. This was confirmed by reanalyzing the set of HPLC-MALDI MS/MS data with no loss in protein identifications, despite the number of virtually executed MS/MS analyses being decreased by 57%.     

Unambiguous identification of volatile organic compounds by proton-transfer reaction mass spectrometry coupled with GC/MS

Interest in on-line measurements of volatile organic compounds (VOCs) is increasing, as sensitive, compact, and affordable direct inlet mass spectrometers are becoming available. Proton-transfer reaction mass spectrometry (PTR-MS) distinguishes itself by its high sensitivity (low ppt range), high time resolution (200 ms), little ionization-induced fragmentation, and ionization efficiency independent of the compound to be analyzed. Yet, PTR-MS has a shortcoming. It is a one-dimensional technique that characterizes compounds only via their mass, which is not sufficient for positive identification. Here, we introduce a technical and analytical extension of PTR-MS, which removes this shortcoming, while preserving its salient and unique features. Combining separation of VOCs by gas chromatography (GC) with simultaneous and parallel detection of the GC effluent by PTR-MS and electron impact MS, an unambiguous interpretation of complex PTR-MS spectra becomes feasible. This novel development is discussed on the basis of characteristic performance parameters, such as resolution, linear range, and detection limit. The recently developed drift tube with a reduced reaction volume is crucial to exploit the full potential of the setup. We illustrate the performance of the novel setup by analyzing a complex food system.     

Automated iterative MS/MS acquisition: a tool for improving efficiency of protein identification using a LC-MALDI MS workflow

We have developed an information-dependent, iterative MS/MS acquisition (IMMA) tool for improving MS/MS efficiency, increasing proteome coverage, and shortening analysis time for high-throughput proteomics applications based on the LC-MALDI MS/MS platform. The underlying principle of IMMA is to limit MS/MS analyses to a subset of molecular ions that are likely to identify a maximum number of proteins. IMMA reduces redundancy of MS/MS analyses by excluding from the precursor ion peak lists proteotypic peptides derived from the already identified proteins and uses a retention time prediction algorithm to limit the degree of false exclusions. It also increases the utilization rate of MS/MS spectra by removing "low value" unidentifiable targets like nonpeptides and peptides carrying large loads of modifications, which are flagged by their "nonpeptide" excess-to-nominal mass ratios. For some samples, IMMA increases the number of identified proteins by ～20-40% when compared to the data dependent methods. IMMA terminates an MS/MS run at the operator-defined point when "costs" (e.g., time of analysis) start to overrun "benefits" (e.g., number of identified proteins), without prior knowledge of sample contents and complexity. To facilitate analysis of closely related samples, IMMA's inclusion list functionality is currently under development.     

Improving protein identification sensitivity by combining MS and MS/MS information for shotgun proteomics using LTQ-Orbitrap high mass accuracy data

We investigated and compared three approaches for shotgun protein identification by combining MS and MS/MS information using LTQ-Orbitrap high mass accuracy data. In the first approach, we employed a unique mass identifier method where MS peaks matched to peptides predicted from proteins identified from an MS/MS database search are first subtracted before using the MS peaks as unique mass identifiers for protein identification. In the second method, we used an accurate mass and time tag method by building a potential mass and retention time database from previous MudPIT analyses. For the third method, we used a peptide mass fingerprinting-like approach in combination with a randomized database for protein identification. We show that we can improve protein identification sensitivity for low-abundance proteins by combining MS and MS/MS information. Furthermore, "one-hit wonders" from MS/MS database searching can be further substantiated by MS information and the approach improves the identification of low-abundance proteins. The advantages and disadvantages for the three approaches are then discussed.     

Peptide electroextraction for direct coupling of in-gel digests with capillary LC-MS/MS for protein identification and sequencing

An electrophoretic method has been developed for the extraction of peptides following in-gel digests of SDS-PAGE separated proteins. During electroextraction, the peptides are trapped on a strong cation-exchange microcartridge, before analysis by capillary LC--ESI-tandem mass spectrometry. The spectra obtained by tandem mass spectrometry are searched directly against a protein database for identification of the protein from which the peptide originated. By minimizing surface exposure of the peptides during electroextraction, a reduction of the detection limits for protein identification is realized. The performance of the peptide electroextraction was compared directly with the standard extraction method for in-gel protein digests, using a standard dilution series of phosphorylase B and carbonic anhydrase, separated by SDS-PAGE. The lowest gel loading in which phosphorylase B was identified using the standard extraction method was 2.5 ng or 25 fmol, and the lowest gel loading in which phosphorylase B was identified using electroextraction was 1.25 ng or 12.5 fmol. The design of the microextraction cartridge allows for direct interfacing with capillary LC, which is crucial for maintaining low detection limits. Furthermore, this method can be used for high-throughput proteomics since it can be easily multiplexed and requires only voltage control and low pressures (approximately 15 psi) for operation. We believe that peptide electroextraction is a significant advance for identification of proteins separated by one-dimensional or two-dimensional gel electrophoresis, as it can be easily automated and requires less protein than conventional methods.     

CE-microreactor-CE-MS/MS for protein analysis

We present a proof-of-principle for a fully automated bottom-up approach to protein characterization. Proteins are first separated by capillary electrophoresis. A pepsin microreactor is incorporated into the distal end of this capillary. Peptides formed in the reactor are transferred to a second capillary, where they are separated by capillary electrophoresis and characterized by mass spectrometry. While peptides generated from one digestion are being separated in the second capillary, the next protein fraction undergoes digestion in the microreactor. The migration time in the first dimension capillary is characteristic of the protein while migration time in the second dimension is characteristic of the peptide. Spot capacity for the two-dimensional separation is 590. A MS/MS analysis of a mixture of cytochrome c and myoglobin generated Mascot MOWSE scores of 107 for cytochrome c and 58 for myoglobin. The sequence coverages were 48% and 22%, respectively.     

Integrated sample processing system involving on-column protein adsorption, sample washing, and enzyme digestion for protein identification by LC-ESI MS/MS

An automated system has been developed for protein identification using mass spectrometry that incorporates sample cleanup, preconcentration, and protein digestion in a single stage. The procedure involves the adsorption of a protein or a protein mixture from solution onto a hydrophobic medium that is contained within a microcolumn. The protein is digested while still bound to the hydrophobic support. The peptides are then eluted from surface digestion to an electrospray ionization mass spectrometer for detection and sequencing. The entire system is fully automated wherein the mass spectrometer is collecting data continuously. We demonstrate that this system is capable of identifying standard protein samples at concentrations down to 100 nM. Further development of this technique may offer a potential solution for proteomics applications that require unattended operation, such as on-line monitoring and identification of microorganisms on the basis of the detection of their protein biomarkers.     

Nanoflow low pressure high peak capacity single dimension LC-MS/MS platform for high-throughput, in-depth analysis of mammalian proteomes

The use of narrow bore LC capillaries operated at ultralow flow rates coupled with mass spectrometry provides a desirable convergence of figures of merit to support high-performance LC-MS/MS analysis. This configuration provides a viable means to achieve in-depth protein sequence coverage while maintaining a high rate of data production. Here we explore potential performance improvements afforded by use of 25 μm × 100 cm columns fabricated with 5 μm diameter reversed phase particles and integrated electrospray emitter tips. These columns achieve a separation peak capacity of ≈750 in a 600-min gradient, with average chromatographic peak widths of less than 1 min. At room temperature, a pressure drop of only ≈1500 psi is sufficient to maintain an effluent flow rate of ≤10 nL/min. Using mouse embryonic stem cells as a model for complex mammalian proteomes, we reproducibly identify over 4000 proteins across duplicate 600 min LC-MS/MS analyses.     

MS for identification of single nucleotide polymorphisms and MS/MS for discrimination of isomeric PCR products

ESI (electrospray ionization) MS and tandem mass spectrometry (MS/MS) were used for the analysis of single nucleotide polymorphisms (SNPs) and more complex genetic variations. Double-stranded (ds) PCR products were studied. PCR products of the proline [5'-x(G17)-x(C38)x-3'] and arginine variants [(5'-x(Gl7)-x(G38)x-3'] of the p53 gene are distinguished by an SNP (cytosine or guanine) and were discriminated using both quadrupole and quadrupole ion trap MS analysis. A 69 bp arginine mutant PCR product [5'-x(C17)-x(G38)x-3'] with a negating switch has the same mass as the proline variant but was readily distinguishable on ion trap MS/MS analysis; fragments containing the mutation site, but not the polymorphism, were identified. The 69 bp PCR products were restriction-enzyme-digested, to create 43 bp fragments. ESI quadrupole ion trap MS/MS analysis of the 43 bp product-ion spectra readily demonstrated both polymorphism and negating switch sites. MS and MS/MS are powerful and complementary techniques for analysis of DNA. MS can readily distinguish SNPs but MS/MS is required to differentiate isomeric PCR products (same nucleotide composition but different sequence).     

Solvent-assisted trypsin digestion of ricin for forensic identification by LC-ESI MS/MS

The castor bean plant (Ricinus communis) is used in large quantities for oil production and is also a common ornamental garden plant. However, the beans contain 1-3% of the highly toxic protein ricin, a type II ribosome-inactivating protein that is covered by the Chemical Weapons Convention, and there have been a number of reports concerning the use, or alleged use, of the toxin in terrorist and criminal activities. In the study reported here, we investigated the potential utility of organic solvent-assisted trypsin digestion of crude extracts containing the closely related toxins ricin or abrin to prepare samples for peptide analysis by liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Diagnostic tryptic fragments of the toxins were detected and unambiguously identified by this procedure. The sample preparation protocol substantially reduces the sample preparation time, from overnight to an hour, and thus greatly reduces the total time required for analyses, to less than 2 h. Furthermore, the reported procedure leaves the disulfide bonds in the protein intact. This is highly relevant in the context of the Chemical Weapons Convention, since the disulfide bond connecting the two chains of ricin indicates the presence of an intact toxin and provides additional forensic evidence for the analytical results.     

Chip-type asymmetrical flow field-flow fractionation channel coupled with mass spectrometry for top-down protein identification

A chip-type design asymmetrical flow field-flow fractionation (AF4) channel has been developed for high-speed separation of proteins and top-down proteomic analysis using online coupled electrospray ionization mass spectrometry (ESI-MS). The new miniaturized AF4 channel was assembled by stacking multilayer thin stainless steel (SS, 1.5 mm each) plates embedded with an SS frit in such a way that the total thickness of the channel assembly was about 6 mm. The efficiency of the miniaturized AF4 channel at different channel lengths was examined with the separation of protein standards by adjusting flow rates in which an identical effective channel flow rate or an identical void time can be maintained at different channels. Detection limit, overloading effect, reproducibility, and influence of channel membrane materials on separation efficiency were investigated. Desalting and purification of proteins achieved during the AF4 operation by the action of an exiting crossflow and the use of aqueous mass-spectrometry-compatible (MS-compatible) buffer were advantageous for online coupling of the chip-type AF4 with ESI-MS. The direct coupling of AF4 and ESI-MS capabilities was demonstrated for the high-speed separation and identification of carbonic anhydrase (29 kDa) and transferrin (78 kDa) by full scan MS and for the first top-down identification of proteins with AF4-ESI-MS-MS using collision-induced fragmentation (CID). The presence of intact dimers (156 kDa) of transferrin was confirmed by AF4-ESI-MS via size separation of the dimers from monomers, followed by multiply charged ion spectral analysis of the dimers and molecular mass determinations. It was also found from these experiments that AF4-ESI-MS analysis of transferrin exhibited an increased signal-to-noise ratio compared to that of direct ESI-MS analysis due to online purification of the protein sample and size separation of dimers with AF4.     

Matrix-assisted laser desorption/ionization coupled with quadrupole/orthogonal acceleration time-of-flight mass spectrometry for protein discovery, identification, and structural analysis

The design and operation of a novel UV-MALDI ionization source on a commercial QqoaTOF mass spectrometer (Applied Biosystem/MDS Sciex QSTAR Pulsar) is described. Samples are loaded on a 96-well target plate, the movement of which is under software control and can be readily automated. Unlike conventional high-energy MALDI-TOF, the ions are produced with low energies (5-10 eV) in a region of relatively low vacuum (8 mTorr). Thus, they are cooled by extensive low-energy collisions before selection in the quadrupole mass analyzer (Q1), potentially giving a quasi-continuous ion beam ideally suited to the oaTOF used for mass analysis of the fragment ions, although ion yields from individual laser shots may vary widely. Ion dissociation is induced by collisions with argon in an rf-only quadrupole cell, giving typical low-energy CID spectra for protonated peptide ions. Ions separated in the oaTOF are registered by a four-anode detector and time-to-digital converter and accumulated in "bins" that are 625 ps wide. Peak shapes depend upon the number of ion counts in adjacent bins. As expected, the accuracy of mass measurement is shown to be dependent upon the number of ions recorded for a particular peak. With internal calibration, mass accuracy better than 10 ppm is attainable for peaks that contain sufficient ions to give well-defined Gaussian profiles. By virtue of its high resolution, capability for accurate mass measurements, and sensitivity in the low-femotomole range, this instrument is ideally suited to protein identification for proteomic applications by generation of peptide tags, manual sequence interpretation, identification of modifications such as phosphorylation, and protein structural elucidation. Unlike the multiply charged ions typical of electrospray ionization, the singly charged MALDI-generated peptide ions show a linear dependence of optimal collision energy upon molecular mass, which is advantageous for automated operation. It is shown that the novel pulsing technique of this instrument that increases the sensitivity for precursor ions scans is applicable to the identification of peptides labeled with isotope-coded affinity tags.     

Infrared multiphoton dissociation and electron-induced dissociation as alternative MS/MS strategies for metabolite identification

A major challenge encountered in mass spectrometric metabolite analysis is the identification and structural characterization of metabolites. Fourier transform ion cyclotron resonance mass spectrometry is a valuable technique for metabolite structural determination because it provides accurate masses and allows for multiple MS/MS fragmentation strategies, including infrared multiphoton dissociation (IRMPD) and electron-induced dissociation (EID). Collision activated dissociation (CAD) is currently the most commonly used MS/MS technique for metabolite structural characterization. In contrast, IRMPD and EID have had very limited, if any, application for metabolite characterization. Here, we explore IRMPD and EID of phosphate-containing metabolites and compare the resulting fragmentation patterns to those of CAD. Our results show that CAD, IRMPD, and EID provide complementary structural information for phosphate-containing metabolites. Overall, CAD provided the most extensive fragmentation for smaller (<600 Da) phosphate-containing metabolites; however, IRMPD generated more extensive fragmentation for larger (>600 Da) phosphate-containing metabolites, particularly for species containing increased numbers of phosphate groups. EID generally provided complementary fragmentation to CAD and showed extensive fragmentation with relatively evenly abundant product ions, regardless of metabolite size. However, EID fragmentation efficiency is lower than those of CAD and IRMPD.     

A four-column parallel chromatography system for isocratic or gradient LC/MS analyses

A novel approach to parallel liquid chromatography/ tandem mass spectrometry (LC/MS/MS) analyses for pharmacokinetic assays and for similar quantitative applications is presented. Modest modifications render a conventional LC/MS system capable of analyzing samples in parallel. These modifications involve the simple incorporation of three valves and four LC columns into a conventional system composed of one binary LC pumping system, one autosampler, and one mass spectrometer. An increase in sample throughput is achieved by staggering injections onto the four columns, allowing the mass spectrometer to continuously analyze the chromatographic window of interest Using this approach, the optimized run time is slightly greater than the sum of the widths of the desired peaks. This parallel chromatography unit can operate under both gradient and isocratic LC conditions. To demonstrate the utility of the system, atorvastatin, five of its metabolites, and their deuterated internal standards (IS) were analyzed using gradient elution chromatography conditions. The results from a prestudy assay evaluation (PSAE) tray of standards and quality control (QC) samples from extracted spiked human plasma are presented. The relative standard deviation and the accuracy of the QC samples did not exceed 8.1% and 9.6%, respectively, which is well within the acceptance criteria of the pharmaceutical industry. For this particular analysis, the parallel chromatography system decreased the overall run time from 4.5 to 1.65 min and, therefore, increased the overall throughput by a factor of 2.7 in comparison to a conventional LC/MS/MS analytical method.